Purification of trhodothyronine



United States Patent PURIFICATION F TRIIODOTHYRONINE John Thomas Plati,Rutherford, and Wilhelm Wenner, Upper Montclair, N. J., assignors toHofimann-La Roche Inc., Nutley, N. 3., a corporation of New Jersey NoDrawing. Application March 10, 1954, Serial No. 415,440

10 Claims. (Cl. 260-519) This invention relates to a method for thepurification of 3,5,3-triiodothyronine.

3,5,3'-triiodothyronine, hereinafter referred to as triiodothyronine, isa substance which occurs, together with thyroxine, in the thyroid gland.These are known substances with thyroid activity. Previous efiorts toobtain triiodothyronine have produced only very small amounts of thedesired compound. When synthetic methods of preparation are employed,10% or more of thyroxine is generally present in the product. By theprocess of this invention triiodothyronine free from thyroxine may nowbe obtained in practical quantities.

According to this invention, triiodothyronine may be purified bycountercurrent solvent extraction employing a water immiscible alcoholand an alkali metal hydroxide as the solvent pair. A solution of thecrude mixture comprising triiodothyronine and thyroxine is introduced atabout the mid-point of a multi-stage solvent extraction system in whichthe water immiscible alcohol and alkali metal hydroxide are incountercurrent flow. The purified triiodothyronine is collected in theheavy solvent which has passed through the system and may be iso latedas a pure compound by precipitation from the alkali metal hydroxidesolution with concentrated hydrochloric acid and subsequentcentrifuging.

The process is carried out by extracting in 30 to 40 stages, preferablyin a continuous countercurrent solvent extraction column which consistsof alternate calming and mixing sections. A column of the type preferredin this process is described in U. S. Patent No. 2,493,265, issuedJanuary 3, 1950, to Edward G. Scheibel.

The light solvent employed in this process is a water immiscible alcoholsuch as a butyl or amyl alcohol, preferably n-butanol. The heavy solventis an alkali metal hydroxide solution, preferably sodium hydroxidesolution. The alkali metal hydroxide is employed at a concentration ofabout 1.5 N to 3 N. The ratio of light solvent to heavy solvent shouldbe maintained within a range of about 1 to 5 to 1 to 10, respectively,on a volumetric basis. Preferably the ratio is maintained at about 1 to7. In a continuous operation, according to the preferred procedure, thedesired ratio is maintained by adjusting the flow rate of the lightsolvent in the column at the flow rate of the heavy solvent.

According to a particularly preferred method of practicing thisinvention, 3,5-diiodothyronine in an aqueous solution of an alkylamine,such as methylamine, is treated with freshly prepared iodine solution atabout room temperature under efficient stirring to iodinate the startingcompound. In order to assure complete iodination, slightly in excess of2 atoms of iodine per mol of diiodothyron-ine is employed. The resultingreaction mixture is distilled at about 60 C. or lower, filtered, anddried.

The crude reaction mixture obtained in this manner, containingpredominantly triiodothyronine and about 10 to thyroxine, is dissolvedin dilute aqueous sodium hydroxide of about 1 to 2 N to form aconcentrated Patented Mar. 5, 1957 solution of the iodinated product inthe alkali (approximately 10 to 17%, preferably 15%, iodination productby weight). Sodium hydroxide solution and butanol are introduced intothe top and bottom, respectively, of a multi-stage, liquid-liquidextraction tower of the type described above until equilibrium isestablished. The flow rate of the n-butanol is maintained at about V;the flow rate of the sodium hydroxide solution.

The solution of iodination product in dilute sodium hydroxide describedabove is slowly introduced at about the mid-point of the extractor. Thecountercurrent flow of light and heavy solvents is continued after allthe feed has been introduced into the extractor until a sample ofalkaline solution withdrawn from the bottom of the column shows no redcolor when treated with hydro chloric acid and sodium nitrite andfinally with ammonium hydroxide. Since a positive test, i. e., theappearance of a red color, indicates that triiodothyronine is present,the failure of the red color to appear in the sample indicates completeextraction of triiodothyronine.

The sodium hydroxide solution obtained from the column is acidified to apH of about 2.5 to 3 with concentrated hydrochloric acid. The flocculentprecipitate which forms upon addition of the acid is the free base,3,5,3'-triiodothyronine, which may be separated by centrifuging or thelike. The free base may be converted into its hydrochloride by treatmentwith an excess of dilute hydrochloric acid, i. e., about 2 N HCl. Thehydrochloride of triiodothyronine may also be prepared directly, withoutseparation of the free base, by adding an excess of hydrochloric acid tothe alkaline solution obtained from the column.

Example A solution of 38.1 grams of powdered iodine and 38.1 grams ofpotassium iodide was prepared by shaking vigorously with suflicientwater to attain a volume of 300 cc. A few particles of iodine remainedundissolved. Titration of a sample of the supernatant liquid with sodiumthiosulfate in the conventional manner showed that the liquid had aniodine concentration of 0.982 N.

A solution of 57.8 grams (0.11 mol) of 3,5-diiodothyronine in 500 cc. of25% methylamine in water was next prepared and 242 cc. (0.24 atom) ofthe iodine solution was added at a uniform rate during 5 /2 hours withefi'icient stirring at 27 C. After stirring for an additional 40minutes, the mixture was distilled at 35 mm. with stirring in a bath at60 to remove the methylamine. The residue was filtered and theprecipitate washed thoroughly with water. The pH of the filtrate andwashings was 8.4. After drying in a desiccator over potassium hydroxide,the crude iodinated mixture containing predominantly3,5,3'-trii-odothyronine melted at 198-199 C. An estimate by paperchromatography indicated that the crude mixture contained l0-l5%thyroxine.

n-Butanol and 2 N sodium hydroxide were shaken together in order toattain an equilibrium between the two solvents. The light and heavylayers were then separated. The light (n-butanol) layer was introducedinto the bottom and the heavy (sodium hydroxide) layer was introducedinto the top of a countercurrent extraction column. The column, 277 cm.long and about 3 cm. in diameter, contained 34 stages, each stage havinga mixing and calming portion. The calming portion of each stagecomprised a roll of wire mesh packing. Paddle type agitators mounted ona rotatable central shaft extended into each of the mixing sections. Theflow rate of the light solvent was maintained at approximately V; theflow rate of the heavy solvent. When equilibrium was attained, 20 gramsof the crude iodinated mixture obtained above dissolved in cc. of 1 Nsodium hydroxidensvas admitted into themiddle of the column in "fourportions duringaboutonehour. The conntercurrentflow of the light andheavy solvents was continued at the same relative rate until 8070 cc. ofthe heavy, aqueous solvent and 1112.00 :cc. int x the slight, :butanolic:solventxwere .coliected. i'FhESlCiIdEOf {the extraction .wasnoted:whenia sample iOfI alkaline L SOILIllOIl'rg-QVBIHO red. :color.uponstreab ment forwalshort timewith hydrochloric acid and: sodiumnitrite :and finally with .ammonium hydroxide.

iThe sodiumhydroxidesolutiont-which waszcollectedwas acidified-withaboutIQQO cc. of concentrated hydrochloric acid. Theztemperature;.rose:'to.;.50 and3,5,3"-triiodothyroninezprecipitatedhas-arilocculent mass. The pH ofthecfilt-ratei wasr21-82. This precipitate was-separatedby centrifugingin :6;portions in .1250 :cc. bottles.

tiiiosconv'ertzthe base into; its hydrochloride, thecontents ofeachzhottlewere treated-separately:byiintroducing into 1300.1cc..:of;1b'oiling 2 :N hydrochloricocid. A :small amount of insolublematerialremained. ;It:was removed by adding 0.5 gram ;of t activatedcharcoal together :with diatomaceous ;earth ,and Jfiltering vthe hotmixture by gravityethroughaafheated funnel. .The-filtrate was thencooled to ;-55. withnstirring;seeded-:andallowed toxstandovernightundi'sturbed. T he .six'individual filtrates yieldedwell-formed;crystals of 3,5;3 -triiodothyronine hydrochloride, M.'P.T20QI202' C., with (ice. (uncorr.). .A second crop ;,'of ;crystals'waslobtained when-the acid filtrates were made :basic with 50%zalkali-topH 5.1 and the resulting precipitate 'wasseparated andcrystallized from 2 .N hydrochloric acid. .Thecompoundis hygroscopic..No evidence-:ofeither diiodothyronine or thyroxine was revealed,hyjpaper chromatography.

13.;51dil0d0thYlOl1iI1B, :3;5,-3-triiodothyronine .and thyroxine occurin :the formof levoand 'dextro stereoisomers: and in I racemic.mixtures. ;The references 1 in the disclosure and the claims tothesescompounds-are applicable to all three forms in which each compoundoccurs.

We claim:

1. A process for the purification of 3,5,3-triiodoth'yronine whichcomprises solvent extracting a solution comprising'3,-5,3-'-triiodothyronine with a Water immiscibleaic'ohol-and an alkalimetal hydroxide in countercurrent flow, collecting theheavy solvent andseparating 3,=5',i3'-triiodothyronine therefrom.

Aproce'ss accordingtoclaim 1 wherein the water immiscible alcohol isn-butanol and-the alkali metal hydroxide l is' sodium hydroxide.

3. LA-proc'e'ss forpurification of 3,5,3-triiodothyronine whichcomprises introducing a solution c0mpri'sing'3,S,'3-

,triiofldothyronine ,into ,a ,multi-stage solvent extractor wherein awater immiscible alcohol and an alkali metal hydroxide are incountercurrent flow, contacting the 3,5,3'-triiodothyronine solutionwith the solvents, with drawing the alkali metal hydroxide solvent fromthe extractor and separating :substantially pure 3,5,3-triiodothyroninetherefrom.

4. A process according'to claim '3 wherein the water immiscible alcoholis n-butanoland the alkalimetal hy+ is sodium hydroxide.

5. -A processaccording torclaimi3 whereinthe 3,5 ,3'- triiodothyronineis separated from the alkali metal hydroxide solvent byprecipitationwith concentrate'd hydrochloric acid. I

6. A process according-4o clai-mB Wherein the alkali metal hydroxide isat a concentration of about 1.5 N to 3 N and the ratio of light solventto heavy solvent is maintained withinthe :rangeof: about 110 5 to 1m-10.

7. A process for the purification Jof 3,5,3'-triiodothyronine freeifrom.thyroxinewhich. comprises dissolving a :crude ;m ix-ture containing3,5,3'.-triiodothyronine in a dilutealkaliametal hydroxide solution,introducing the alkali:metal:hydroxideesolution of 3,5,3'-triiodothyronine at :about the. mid-:pointof a-multi-stage solventextraction columniwherein talightsolvent. comprising n-butanol and aheavysolventcomprising dilute sodium hydroxide are incountercurrent.flow, continuously contacting the feed mixtureywithxhe,countercurrentlyflowing solvents, continuously withdrawing;and;collecting sodiumhydroxide solution from :the extractor, .and precipitating substantially.pure ;3;, 5.,'3 -triiodothyronine .from the sodiumhydroxide ssolution.

8..A;.process;.according toclaim 7 whereinthe 3,5,3 triiodothyronine isconverted toits hydrochloride salt by u separating;the;3;5.,3i-triiodothyronine.- from'the sodium hydroxide solution and treating the3 ,5 ,3'-triiod0thyronine with an excess of 1 dilute-hydrochloric acid.

19. .A process accordingto .claim 7 wherein the. light andhcavyisolventsareina ratio of .1 to 7, respectively.

10. Aprocess according toclaim 9 wherein thealkali metal hydroxidesolventpfor thecrude mixtureis 1.to 2 N sodium hydroxide and the heavyextracting. solvent is about 2 .N .sodiumhydroxide.

References Cited in the filerof thispatent .Gross et. al.: 'Biochem.Journal, .vol 53, pp. 645- (1953).

1. A PROCESS FOR THE PURIFICATION OF 3,5,3''-TRIIODOTHYRONINE WHICHCOMPRISES SOLVENT EXTRACTING A SOLUTION COMPRISING3,5,3''-TRIIODOTHYRONINE WITH A WATER IMMISCIBLE ALCOHOL AND AN ALKALIMETAL HYDROXIDE IN COUNTER CURRENT FLOW, COLLECTING THE HEAVY SOLVENTAND SEPARATING 3,5,3''-TRIIODOTHYRONINE THEREFROM.